This project utilizes NMR spectroscopy to study the molecular components of HIV and model systems. Recent studies have focused on: 1) analysis of the solution conformation and dynamics of the ribonuclease H (RNase H) domain of HIV reverse transcriptase, and the characterization of substrate-induced active site formation; 2) understanding the protein-mediated interactions of RT substrates and inhibitors; 3) understanding the complex conformational maturation process that converts the initial p66 protein into the p66/p66' homodimer, and finally the p66/p51 heterodimer; 4) understanding the basis for the subunit-dependent elimination of one RH domain 5) identification of potential targets that would interfere with the maturation process. Project 1. During the past year we have continued our studies of the maturation pathway of HIV-1 reverse transcriptase. In order to achieve economy of the coding sequence requirements, RT utilizes a metamorphic polymerase domain that is able to adopt two alternate structures and thus fulfill two different functions. The domain containing the active fold forms the active polymerase site in the p66 subunit, while the domain that adopts an inactive form fulfills a structural requirement of the p51 subunit, stabilizing the RT p66/p51 heterodimer. We have continued to investigate the metamorphic transition and the factors that influence it. The C-terminal residues of the palm domain, F227-V241, are partially disordered in the monomer, but form a three stranded beta-sheet in the p66 subunit. This transition is driven by the presence of two factors: residues at the turn positions that intrinsically favor beta-turn structures, and hydrophobic residues that are positioned to reinforce the structure of the beta-sheet. These intrinsic local preferences become important when the monomer domains dissociate, and facilitate the metamorphic transition. Assignment of RT216 was hindered by the tendency of the isolated construct to aggregate. Based on the observed shifts, this aggregation appears to involve primarily residues on the palm domain beta-sheet that are solvent exposed because the construct lacks the C-terminal residues of the palm domain that cover this hydrophobic region of the structure in the p66 subunit. For this reason, resonance assignments required triple labeling with H-2, C-13, and N-15, and were also facilitated by several studies in which specific residue types were labeled separately. The assignments have been deposited in the BioMagResBank. Project 2. One of the central questions of RT maturation involves the partial processing of the p66 precursor to form the p51 subunit of RT. Since each RT molecule contains a p66 and a p51 subunit, this requires that exactly 50% of the molecules be processed. The simplest way to satisfy this constraint is to form an asymmetric p66/p66 homodimer in which structural differences between the two subunits lead to subunit-specific processing. Based on a previous series of studies, we proposed that the metamorphic transformation occurs prior to dimerization, leading to formation of the asymmetric homodimer. In the asymmetric homodimer p66 chain destined to become truncated, a tug-of-war exists between the polymerase domain in the inactive fold, and the RH domain, since the polymerase C-terminus and the RH N-terminus contain common residues. This does not occur in the active subunit, since there is no conflict between the two domains for common residues. This analysis leads to subunit-selective unfolding of a single RH domain. It was recently noted that expression of the isolated RH domain in bacterial systems leads to the co-expression of both RH monomers and dimers. During the past year, we structurally characterized the dimer as a domain-swapped structure. Domain swapped dimers occur when pairs of partially unfolded molecules encounter each other and can form a set of complementary interactions that stabilize the same overall fold as a dimer. Dimer formation tends to be driven in part by loops that are two short to link specific structures in the monomer can more effectively link the two structures in the dimer. This is exactly what happens to the RH domain. In the RH domain structures, the loop connecting helices B and D is unfavorably short. In the domain swapped structure, the loop becomes a hinge that connects extended helices B and D. The interaction of the two helices is mediated by the domain N-terminal Tyr427. Thus, transfer of this residue from the RH to the polymerase domain facilitates RH domain unfolding, consistent with the previously proposed tug-of-war model. Project 3. Based on the subunit-specific RH domain unfolding model summarized above, stabilizing the supernumerary RH domain on one p66 subunit represents one approach to inhibiting formation of the mature heterodimer. In principle, RH domain active site-targeted inhibitors would be expected to interfere with RH unfolding and hence to retard or block RT maturation. In our recent studies, we used Ile-labeled p66/p66 homodimer to follow RT maturation and subunit-specific RH domain unfolding. The resonances off Ile434 near the RH domain N-terminus provide a particularly convenient way of studying this, since separate resonances are observed for the domain on the two subunits of the asymmetric homodimer. We found that addition of an active site-directed isoquinolone ligand - 2-hydroxyisoquinoline-1,3(2H,4H)-dione (HIQ) produces a significant reduction in the rate of disappearance of the RH domain isoleucine resonances in the subunit in which the polymerase domain has the inactive fold. Thus, the Mg-HIQ significantly interfered with RH unfolding and with maturation of the connection domain on one p66 subunit. Thus, it is possible to retard RT maturation with agents that stabilize the supernumerary RH domain. This demonstrates a new approach for inhibiting RT activity based on interfering with the RT structural maturation process.